Diseño de la secuencia didáctica (sd)

5.6.1 Import of radioactively labelled proteins into intact chloroplasts

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S-labelled precursor proteins (translation products) in the maximal amount of 10% (v/v) in the reaction were mixed with freshly prepared intact pea chloroplasts (equivalent to 15-20 µg chlorophyll) in import buffer (330 mM sorbit, 50 mM HEPES/KOH pH 7.6, 3 mM MgSO4,

10 mM Met, 10 mM Cys, 20 mM K-gluconate, 10 mM NaHCO3, 2% BSA (w/v)) and up to 3

mM ATP in a final volume of 100 µl (Waegemann and Soll, 1995). Optionally 80 mM KiPO4

were added to enhance import rate (Hirsch and Soll, 1995). The import mix was incubated at 25°C for up to 20 minutes, depending on experimental requirements. Chloroplasts were reisolated over a 40% Percoll cushion, washed, and samples were separated by SDS-PAGE. Resulting gels were fluorographed (Bonner and Laskey, 1974) if needed, dried and laid on x-ray sensitive films over night.

5.6.1.1 Pulse-chase experiments

During import into chloroplasts it is possible to track changes in localization and quantity of a protein at different times, to the point it reaches its final destination. For this purpose radioactively labelled precursor protein was added to chloroplasts corresponding to 20 µg chlorophyll in the import mixture without ATP (see chapter 5.6.1) and to final volume of

100 µl. For MGD1 80 mM KiPO4 were added to enhance import. Samples prepared this way

were incubated for 2 minutes on ice to accomplish the binding to the import receptors on the chloroplast surface. Samples were centrifuged at 1500xg for 1 minute, washed once in the import buffer and the final pellet was resuspended in import buffer containing 3 mM ATP to allow complete import. The import reactions were performed from 0 up to 32 minutes at 25ºC. Reactions were stopped after different times by addition of Laemmli buffer and samples were analyzed by SDS-PAGE.

For chasing the soluble stromal intermediates, Tic110 and pS-110N-SSU were incubated with 20 µg chloroplasts in the import mix containing 3 mM ATP for 2 minutes at 25ºC. Chloroplasts were pelleted and resuspended in the new import mix without ATP, containing 0.5 µg thermolysin per µg chlorophyll. The reaction was kept on ice for 10 minutes. Afterwards, the chloroplasts were reisolated on a Percoll cushion and resuspended in the import mix in the presence or the absence of 3 mM ATP. Samples were kept for 5 minutes on ice and imported for 10 minutes at 25ºC. All described steps were performed in the dark to minimize the generation of internal ATP, produced inside chloroplasts via photophosphorylation process. After import reaction, chloroplasts were pelleted 1 min at 1500xg, washed and separated into soluble and membrane fractions (section 5.6.2.1).

For the purpose of tracking the energy requirement of Tic110 and pS-110N-SSU on reexport import pathway radioactively labelled precursors were incubated with 20 µg chloroplasts in the import mix containing 3 mM ATP for 2 minutes at 25ºC. Afterwards chloroplasts were pelleted and resuspended in the new import mix containing either ATP, GTP or PEP at the final concentration of 3 mM, or non-hydrolyzable ATP homologs AMP- PNP and/or ATP--S, at the final concentration of 5 mM. Fresh import mix was incubated on ice for 5 minutes and subsequent 10 minutes on 25ºC. All incubations were performed in the dark to diminish the influence of internally produced ATP. Afterwards, chloroplasts were pelleted 1 min at 1500xg, washed, and separated into soluble and membrane fractions.

5.6.1.2 Competition with pOE33 and mOE33 proteins

Up to 10 µM of purified competitor protein pOE33, as well as its mature form mOE33 (see section 5.10.3) were added to the import mixture. The import experiment was performed as described in section 5.6.1. Maximum 15 µg of chlorophyll per reaction was used and the import reaction lasted 5 (pSSU) to 10 or 12 minutes (Tic22, MGD1) at 25°C.

5 Methods

5.6.1.3 Competition for import by the cytosolic domain of Toc34 receptor GTPase

In addition to 3 mM ATP and 3 mM GTP, up to 10 µM Toc34∆TM were added to the import mixture. First, Toc34∆TM was preincubated with GTP in the import mixture for 10 minutes on ice. Subsequently, radioactively labelled translation product has been added for another 10 minutes to allow the interaction of preprotein with Toc34∆TM. At the end, 15 µg of chloroplasts were added for import and the reaction was performed for 10-12 minutes at 25°C for Tic22 and MGD1 and 5 minutes for pSSU.

5.6.1.4 Inhibition of import by Ni2+ ions

For the purpose of stopping the import reaction on the level of the outer envelope binding 1 mM NiSO4 was added to the import mix (Rothen et al., 1997). Ni2+ ions interact with the

His-tag on the C-terminus of radioactively translated proteins and inhibit their passage across the outer envelope. The alternative to allow only binding, but not the import of preproteins into chloroplasts, was the incubation of the import mixture for 5 minutes on ice.

5.6.1.5 Protease posttreatment of intact chloroplasts

After import, the progress of translocation of proteins through the outer chloroplast envelope was controlled by the treatment of intact organelles with the protease thermolysin. Chloroplasts were pelleted from the import reaction at 1500xg for 1 minute at 4°C and resuspended in 100 µl digestion buffer (330 mM sorbit, 50 mM HEPES/KOH, pH 7.6, 0.5 mM CaCl2). The digestion started with the addition of thermolysin (0.5 µg per µg

chlorophyll) and was incubated on ice for 20 minutes. The reaction was stopped by addition of 5 mM EDTA, chloroplasts were pelleted again and washed in the digestion buffer containing 5 mM EDTA.

5.6.2 Suborganellar localization of imported constructs

5.6.2.1 Chloroplast fractionation into soluble and insoluble fractions

To distinguish between integral membrane proteins and soluble or peripheral membrane proteins, chloroplasts reisolated after import were lysed in 10 mM HEPES/KOH pH 7.6 for 30 minutes on ice, followed by centrifugation at 265,000xg for 10 minutes at 4°C to separate the membranes from a soluble fraction.

5.6.2.2 Extraction of proteins with 6M Urea and 0.1 M Na2CO3 after import

After import, reisolated chloroplast were lysed (see section 5.6.2.1), pelleted at 256,000xg, and the pellet was subsequently treated with 6M urea in 10 mM HEPES/KOH pH 7.6 or 0.1 M Na2CO3 pH 11.5 for 20 minutes at RT. Samples were centrifuged at 256,000xg for 10

minutes at 4°C and the pellet and soluble fractions were analyzed by SDS-PAGE.

5.6.2.3 Chemical crosslinking and immunoprecipitation

After import, chloroplasts were re-isolated on a Percoll cushion, washed and chemical crosslinking was performed by incubation of chloroplasts with 0.5 mM dithiobis-succinimdyl- proprionate (DSP) in 330 mM sorbit, 50 mM HEPES/KOH pH 7.6, 0.5 mM CaCl2, for 15

minutes at 4ºC. The reaction was stopped by the addition of 125 mM glycin and further incubation at 4ºC for 15 minutes. Chloroplasts were washed twice in 330 mM sorbit, 50 mM HEPES/KOH pH 7.6, 0.5 mM CaCl2 and finally lysed in hypotonic buffer (20 mM

HEPES/KOH pH 7.6, 5 mM EDTA) for 30 minutes on ice. A total membrane fraction was recovered by centrifugation at 256,000xg for 30 minutes. Membranes were solublized in 1% SDS (w/v), 25 mM HEPES/KOH pH 7.6, 150 mM NaCl, diluted tenfold in the above buffer in the absence of SDS, centrifuged for 2 minutes at 20,000xg and the supernatant was used for immunoprecipitation with the antisera against Toc75(III), Toc75(V), Toc34, Tic110 and OEP16. Antisera for the previously indicated proteins were incubated with membranes and 0.5% egg albumine, rotating for 1 hour at RT, followed by purification on Protein A- Sepharose. The affinity matrix was washed 3 times with 10 bead-volumes of the mentioned buffer before the elution with Laemmli sample buffer in the presence of β-mercaptoethanol to split the crosslink products.